Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

TLDR

Spheroids provide a 3‑D in‑vitro model for tumor biology, but existing generation methods suffer from size heterogeneity, long culture times, and limited throughput. The study introduces a rapid protocol for producing single spheroids in suspension culture within individual wells. By seeding 1,000–20,000 cells into poly‑HEMA‑coated 96‑well plates, centrifuging at 1,000 g for 10 min, and adding small amounts of Matrigel, the authors generate homogeneous, compact spheroids in 24 h and screen media additives to convert loose aggregates into spheroids. The protocol yields compact, single spheroids from 20 diverse tumor cell lines within 24 h, enabling high‑throughput functional and toxicity analyses.

Abstract

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.